The goal of this project is to develop effective molecularly-defined immune adjuvants for active immunization against cancer, and with direct relevance for more potent immune adjuvants for immunization against infectious pathogens. Although tolerance to cancer is reversible, immunization against cancer self-antigens faces substantial hurdles related to tolerance and immune regulation. Potent immune adjuvants will be necessary for successful active immunization against cancer self-antigens, which are inherently poorly immunogenic. Recently, we have acquired data that demonstrates robust activation of cells from the adaptive immune response in animals immunized with gene-fusion adjuvants. We use genetic adjuvants (plasmid DNA) that combine microbial genes, including VP22 (Herpesvirus) and Exotoxin A (Pseudomonas aeruginosa), with an optimized self-antigen (tyrosinase-related protein 1 = TYRP1) to generate fusion gene products that potentiate a synergistic and highly effectual immune response. DNA encoding a variety of candidate microbial gene-fusions have been carefully selected based on extensive preliminary screening and on our understanding of how these molecules regulate the activation of acquired immune cells. The microbial gene-fusion adjuvants that elicit the most potent activation of immune cells will be combined onto a single plasmid, linked by an 18 amino acid "2A sequence" from the foot-and-mouth disease virus (FMDV), to generate a bicistronic DNA vaccine. Specific Aim 1 examines whether combining two or more gene-fusions coupled by the 2A sequence can increase the level of immune cell activation compared to a single gene-fusion. Specific Aim 2 studies the mechanism of T-cell activation from the most potent 2A-linked gene-fusion chimeras. Specific Aim 3 evaluates whether multi-copy DNA microbial fusion vaccines are effective, as combinatorial agents that can be applied with an IL-12/Fc fusion DNA construct against different tumors. [unreadable] [unreadable] [unreadable]